NEW BASE-ALTERED ADENOSINE ANALOGUES: SYNTHESIS AND AFFINITY AT ADENOSINE A₁ and A_2A RECEPTORS

Abstract

N⁶-Substituted adenosine analogues containing cyclic hydrazines or chiral hydroxy (ar)alkyl groups, designed to interact with the S2 and S3 receptor subregions, have been synthesized and their binding to the adenosine A₁ and A_2A receptors have been investigated. Examples of both types of compounds were found to exhibit highly selective binding (K_i in low nM range) to the rat A₁ receptor.

Introduction

The physiological functions of adenosine have been extensively studied in recent years. Adenosine exerts its biological effects via extracellular purinergic receptors, termed A₁, A_2A, A_2B, and A₃, which are distributed throughout a wide variety of tissues in mammalian systems.^1–3 Although adenosine has been approved for clinical use by the FDA for the treatment of supraventricular tachycardia, its therapeutic application is limited by its rapid metabolic inactivation and its nonselectivity for the receptor subtypes.⁴ There has been considerable interest in the development of adenosine receptor agonists that mimic the pharmacological properties of adenosine but with greater metabolic stability and with higher receptor specificity.^5–7 Adenosine agonists with high A₁ or A_2A receptor selectivity are of potential interest as antihypertensives, antiarrhythmics, analgesics, antipsychotics, and anticonvulsants.^8,9 Several recent reports of highly potent and selective adenosine A₁ or A_2A receptor agonists have focused attention on strategic modifications at the N⁶-, C²-, and 5′-modified adenosines.^5–7,10

However, there have been very few adenosine analogues where a N-N bond exists at the purine 6-position. 6-Hydrazinopurine riboside¹¹ has receptor affinity in the micromolar range (K_i values for A₁ = 29.7 μM, A₂ = 7.3 μM) but 2-chloro-N⁶-[4-(phenylthio)-1-piperidinyl] adenosine¹² exhibits strong A₁ receptor binding and A₁ to A₂ receptor selectivity (A₁ K_i = 0.9 nM, A₂ K_i = 470 nM, A₂/A₁ ratio = 522). The nitrogen isostere of CPA, N6-(1-pyrrolidinyl)adenosine has been reported by us as a potent and selective A₁ agonist (K_i = 8.0 nM for A₁ and 2800 nM for A₂ and a selectivity ratio A₂/A₁ of 350).¹⁰

The model of the N⁶-region of the A₁ receptor has been derived from the structure of (R)-PIA and is based on the assumption that each single part of the C6 substituent (N⁶, C¹, C², C³ and phenyl) positively contributes to the affinity.^13,14 Each of these parts corresponds to a receptor subregion, termed N⁶, S1, S2, S3, and aryl. The chirality at the C² carbon, occupying the S2 subregion, produces a high degree of stereoselectivity for R- vs. S- isomers. N⁶-[(S)-1-Hydroxy-3-phenyl-2-propyl]adenosine, which has a hydroxyl group on the C³ carbon corresponding to the S3 subregion, retains high affinity and selectivity for the A₁ receptor (A₁ K_i = 2.7 nM, A₂ K_i = 390 nM, and a selectivity ratio A₂/A₁ of 144).¹³ This paper reports on the design, synthesis, and adenosine receptor binding studies of new N⁶-substituted adenosine analogues containing cyclic hydrazines or chiral hydroxy (ar)alkyl groups.

Chemistry

Adenosine was used as the starting material in the synthesis of N⁶-substituted adenosine analogues (3–9). It was acetylated with acetic anhydride, 4-dimethylaminopyridine, triethylamine in acetonitrile at 60 °C (Scheme 1).¹⁵ 2′,3′,5′-Tri-O-acetyladenosine was converted to the 6-iodo compound 1 by a thermally-induced radical deamination-halogenation reaction with n-pentyl nitrite and diiodomethane in acetonitrile at 60 °C.¹⁶ The 6-iodo compound 1 was treated with cyclic hydrazines or chiral (ar)alkylamines in the presence of triethylamine in N,N-dimethylformamide or chloroform/ethanol at 60 °C to provide the N⁶-substituted triacetates 2a–g, which were subsequently deprotected with sodium methoxide in anhydrous methanol or anhydrous ammonia in absolute ethanol to afford target compounds 3–9.

Scheme 1.

Guanosine served as the starting material in the synthesis of 2-chloro-N⁶-substituted adenosine analogues (13–18) (Scheme 2). It was acetylated by the same procedure that was used for adenosine but at ambient temperatures followed by reaction with phosphorus oxychloride and N,N-diethylaniline at 60 °C to give 2-amino-6-chloro compound 10 in 87% yield (Scheme 2).¹⁷ The key intermediate, the 2,6-dichloro compound 11, was prepared by the radical deamination-halogenation reaction of 2-amino-6-chloropurine riboside 10 in the presence of n-pentyl nitrite and excess carbon tetrachloride.¹⁶ The 6-chloro group of 11 was selectively displaced by the cyclic hydrazines or chiral (ar)alkylamines in the presence of triethylamine in DMF or chloroform/ethanol at 60 °C by taking advantage of the greater nucleophilic lability of the 6-position compared to the 2-position. Subsequent deprotection with sodium methoxide in methanol or anhydrous ammonia in absolute ethanol afforded the target compounds 13–18. 2-Iodo-N⁶-substituted adenosine analogues (21–25) were prepared via the key intermediate, the 6-chloro-2-iodo compound 19, by the same synthetic methodology used for the synthesis of the 2-chloro compounds (Scheme 3).

Scheme 2.

Scheme 3.

Results and Discussion

The A₁ receptor affinity and the A_2A/A₁ selectivity of some chosen analogues were carried out with rat brain or striatal membranes using radioligand binding assays (Table 1). The high A1 affinity and selectivity of N⁶-(1-pyrrolidinyl)adenosine (3), the corresponding N⁶-(1-piperidinyl) adenosine (4) and its 2-chloro analogue 14, and the 2-chloro N⁶-(1-morpholino)adenosine (16) with K_i values for the A₁ receptor of 7.3, 3.6, 4.9, and 8 nM, respectively, and with A_2A values being in the μM range or higher are of interest. These results, when compared to the low affinity of 6-hydrazinopurine riboside (K_i for A₁ = 29.7 ±6.6 μM, K_i for A_2a = 7.34 ± 1.12 μM),¹¹ suggest that the destabilizing effects of the polar hydrazino functionality can be offset by stabilizing interactions of larger N⁶-substituents whose additional carbons interact with the distal hydrophobic N⁶-subregion. The morpholino analogue 6 is >2000-fold less potent at A₁ receptors than the piperidinyl analogue, 4. Thus, the distal ether functionality destabilizes the binding to the receptor. This destabilization can be overcome by adding a 2-chloro substituent as in 16. The chiral compounds synthesized are expected to have interaction with a number of N⁶-subregions (S1, S2, S3, and aryl) and can be used as probes to study spatial and stereochemical requirements, especially in the S2 receptor subregion. For example, while the (S) and (R) isomers of N⁶-(1-hydroxy-4-methyl-2-pentyl)adenosines 8 and 9 showed low A₁ binding affinity (139 and 224 nM, respectively) and poor A_2A affinity (mM range), dramatic differences are seen in the affinities of the (S) and (R) isomers of N⁶-(1-hydroxy-3-phenyl-2-propyl)adenosines. For example, in the case of 17 and 18, the compound with (S) chirality of the N⁶-substituent is a factor of about 200 times more potent than the corresponding (R)-isomer at A₁ receptors. There is also much greater selectivity between A₁ and A_2A binding for the (S) compared to the (R) isomer. Related results were obtained for the 2-iodo compounds 24 and 25. The 2-iodo analogues (21, 23, 24, 25) were each less potent at both A₁ and A_2A receptors than the corresponding 2-chloro analogues (14, 16, 17, 18). Curiously, for the piperidinyl analogues, a 2-iodo group (21) diminished potency at A₁ receptors versus the 2-H analogue (4), while for the morpholino analogues, a 2-iodo group (23) enhanced A₁ potency versus the 2-unsubstituted compound (6). At the A_2A receptors, introduction of a 2-halo group resulted in enhanced potency compared to the 2-H case. The target compounds are stable with respect to deamination by mammalian adenosine deaminase. They are not expected to be substrates for cellular kinases.

Table 1.

Affinities of Selected Adenosine Analogues in Radioligand Binding Assays at A₁ and A_2A Receptors ^a,^b

Compound No.	A1	A2A
3	8.0	280010
4	7.30 ± 1.25	29% at 10−4 M
6	15,500 ± 1900	36±7 % at 10−4 M
8	139 ± 44	<10% at 10−5 M
9	224 ± 48	30,300±14,800
14	3.55 ± 0.35	936 ± 237
16	4.89 ± 0.27	1900 ±520
17	2.41 ± 0.46	492 ± 87
18	452 ± 65	10,400 ± 3600
21	71.0 ± 23.4	8630 ± 2220
23	113 ± 35	19,400 ±7000
24	23.0 ± 8.0	1360 ± 310
25	989 ± 105	49 ± 2% at 10−4 M

^aDisplacement of specific [³H](R)-PIA binding in rat brain membranes, expressed as K_i ± S.E.M. in nM (n = 3–6), or % of displacement at indicated conc.

^bDisplacement of specific [³H]CGS 21680 binding in rat striatal membranes, expressed as K_i ± S.E.M. in nM (n = 3–6), or % of displacement at indicated conc. Radioligand Binding Assays. For all binding experiments, adenosine deaminase was present (3 IU/mL) during the incubation with radioligand. [³H]CGS 21680 binding to striatal A_2A-receptors in rat brain was carried out as described¹⁸ using 20 μM 2-chloroadenosine to determine nonspecific binding. The binding of [³H]R-PIA to rat cortical A₁-receptors was carried out as previously described.¹⁹ For competition studies, IC₅₀ values were determined using the Inplot computer program (Graphpad, San Diego, CA) and converted to apparent K_i values using K_D values and the Cheng–Prusoff equation.²⁰ K_D values in rat brain for [³H]PIA and [³H]CGS 21680 binding were 1.0 and 15 nM, respectively, at A₁ and A_2A receptors. Concentrations of [³H]PIA and [³H]CGS 21680 used in competition experiments were 1.0 and 5.0 nM, respectively.

In conclusion, N⁶-substituted adenosine analogues containing cyclic hydrazines or chiral hydroxy (ar)alkyl groups, designed to interact with the S2 and S3 receptor subregions, have been synthesized. Affinity studies of selected analogues to the adenosine A₁ and A_2A receptors were carried out with rat brain or striatal membranes using radioligand binding assays. Both types of compounds investigated were found to exhibit highly selective binding to the A₁ receptor (A₁ K_i = low nM range, A_2A K_i = > μM range). For pairs of diastereoisomers, the (S)-isomer was significantly more potent than the (R)-isomer. Interestingly, the (S)-isomers of 8, 17, and 24 resemble more closely the structure of (R)-PIA (more active isomer) than (S)-PIA (less active isomer) in terms of alignment of the -CH₃ of PIA compared to the -CH₂OH of 8, 17 and 24.